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The Distribution of Innate Antioxidant Enzymes in Rat Cochleas

Other Title
쥐 와우에서의 내인성 항산화효소의 분포
Authors
이, 해경
Degree
Master (2011)
Abstract
Background and Purpose: Gentamicin (GM) is a widely used aminoglycoside antibiotic. However, the clinical utility of aminoglycosides is limited by the potential for ototoxicity and nephrotoxicity. In the cases of aminoglycosides’ ototoxicity, the damage of outer hair cells (OHCs) progresses in a base-to-apex gradient. Although the mechanism of GM ototoxicity has not been identified with certainty, the generation of highly reactive oxygen species (hROS) appears to be an important and early step in GM-induced HC death. The differential sensitivity of basal, middle, and apical turn HCs appears to be related to differences in hROS within HCs. Similarly, the higher sensitivity of the first, second and then third row of OHCs, followed by IHCs, to GM damage was preceded by a similar pattern of hROS formation. The differential vulnerability across cochlear turns and HC rows in GM-induced HC damage may be caused by following reasons. First, differential innate resistance of HCs to aminoglycoside may be related with this phenomenon. That is, apical turn HCs may have lots of innate antioxidant enzymes such as glutathione (GSH), superoxide dismutase (SOD), or catalase, compared to those in basal turn HCs. The only one English paper was reported relating with this hypothesis until now. Sha, et al. reported that the dose of GSH was detected two-fold more in apical turn HCs than that in basal turn HCs in in vitro study using chicken cochlears. Second, the uptake or distribution of GM may be different among cochlear turns or rows of HCs. Unfortunately, the data about this hypothesis has not, however, been reported so far. The purposes of the present study were to evaluate the distribution of innate antioxidant enzymes including GSH and SODs in organ of Cortis using in vivo animal studies and to identify the change of expression of antioxidant enzymes in in vitro studies.

Materials and Methods: Sprague-Dawley rat (250g, 6weeks) were classified into two groups, the control group and GM group (GM 160mg/kg, Intraperitoneal injection for 10 days). After cryo-fixation of cochleas, immunostaining for expression of GSH, Cu/Zn-SOD (SOD1), and Mn-SOD (SOD2) was performed. The cochleas were co-labeled with Phalloidin (hair cell markers) and DAPI (nucleus detection). The intensity of antioxidant expression was quantitatively evaluated along cochlear turns and hair cell rows using ImageJ?? program. HEI-OC1 cells were cultured and treated with 0.4mM GM for 48h. These cells were immunostained with antibodies against GSH, Cu/Zn-SOD, or Mn-SOD with GM antibodies. The expression of antioxidant enzymes was evaluated with a fluorescence microscope.

Results: The expression of GSH, Cu/Zn-SOD, and Mn-SOD was higher in IHCs than OHCs in normal rats. However, Mn-SOD was highly detected in Deiter’s cells than HCs. For GSH, Cu/Zn-SOD, inner pillar cells also showed strong expression like that of IHC. The expression pattern of antioxidant enzymes in cochleas in GM-treated rats was similar to that in control rats. Contrary to previous hypothesis, basal turn OHCs showed stronger intensity of GSH, Cu/Zn-SOD, and Mn-SOD than middle and apical turn OHCs. IHCs showed similar fluorescence intensity of antioxidant enzymes along cochlear turns unlike OHCs. In in vitro study, HEI-OC1 cells treated with GM showed increased expression of GSH, Cu/Zn-SOD, and Mn-SOD compared to control cells.

Conclusions: Innate antioxidant enzymes including GSH, SOD1 and SOD2 were distributed much more in basal turn HCs than apical turns in rat cochlear turns unlike previous hypothesis. IHCs showed higher expression of antioxidant enzymes than OHCs and this finding was well related with relative higher resistant characteristics of IHC to toxic stimuli. When rat cochleas or auditory cells were exposed to GM, the expression of antioxidant enzymes was up-regulated in both in vivo and in vitro studies. The differential vulnerability across cochlear turns in GM-induced HC damage may be not directly related with the distribution of innate antioxidant enzymes in HCs.
Keywords
와우내인성항산화효소RatCochleasInnate Antioxidant Enzymes
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Theses > Graduate School of Biomedical Sciences > Master
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이, 해경
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